The liver represents one of the most important targets for RNA interference-mediated gene knockdown therapies, yet a safe and efficacious non-viral liver-targeted gene knockdown strategy has yet to emerge. This is primarily due to the lack of delivery vehicles with well controlled size and shape, and high colloidal and complex stability in physiological media and in endocytic compartments while maintaining efficient release of siRNA in the cytosol. The overall objective of this proposed study is to develop an efficient method to facilitate controlled condensation of siRNA and prepare siRNA-compacting nanoparticles with tunable shapes by assembling with polycation-PEG copolymers and varying solvent polarity, and to test the hypothesis that the shape of siRNA nanoparticles significantly influences their cellular uptake, biodistribution and gene knockdown efficiency in vitro and in vivo through retrograde intrabiliary infusion. This study is built on our recent findings that siRNA-compacting nanoparticle with distinct shapes (spherical, rod-like and worm-like) can be prepared by condensing siRNA with polycation-PEG copolymers and by tuning solvent polarity; and the intrabiliary infusion is an effective administrative route for delivering siRNA nanoparticles to th liver. With this Exploratory Grant, we plan to (1) determine the key parameters that control the shape and size of copolymer/siRNA nanoparticles, and optimize protocol to achieve high stability in extracellular environment while maintaining efficient release of siRNA in the cytosol; and (2) investigate shape dependent cellular uptake and knockdown efficiency for siRNA micellar nanoparticles in vitro, and demonstrate efficient delivery and high gene knockdown efficiency of shaped siRNA nanoparticles in rat liver by intrabiliary infusion. This study will provide an enabling technology for controlling the shape of RNA-compacting nanoparticles, demonstrate the effectiveness of intrabiliary infusion for liver-targeted delivery of siRNA nanoparticles, and reveal the shape dependence in nanoparticle-mediated gene knockdown in the liver and in vivo biodistribution. It will provide key insights into designing more efficient delivery strategies for liver-targeted RNA therapeutics. PUBLIC HEALTH RELEVANCE: This study will provide an enabling technology for controlling the shape and size of RNAcompacting nanoparticles that mimic natural virus particles and demonstrate the effectiveness of intrabiliary infusion for liver-targeted delivery of siRNA nanoparticles. It will reveal how siRNA nanoparticle shape influences their nanoparticle transport and gene knockdown efficiency in the liver and offer key insights into designing more efficient delivery strategies for RNA therapeutics for treating a variety of liver-specific disease.